Isocitrate dehydrogenase phosphorylation

The serine residue of isocitrate dehydrogenase that is phos-phorylated by protein kinase lies within the active site of the enzyme. This situation contrasts with most other examples of covalent modification by protein phosphorylation, where the phosphorylation occurs at a site remote from the active site. What direct effect do you think such active-site phosphorylation might have on the catalytic activity of isocitrate dehydrogenase (See Barford, D., 1991. Molecular mechanisms for the control of enzymic activity by protein phosphorylation. Bioehimiea et Biophysiea Acta 1133 55-62.)... 

D. C. LaPorte and D. E. Koshland, Jr., Phosphorylation of isocitrate dehydrogenase as a demonstration of enhaced sensitivity in covalent regulation. Nature, 305, 286-290 (1983). 

In the presence of adequate O, the rate of oxidative phosphorylation is dependent on the availability of ADR. The concentrations of ADR and ATR are reciprocally related an accumulation of ADR is accompanied by a decrease in ATR and the amount of energy available to the celL Therefore, ADR accumulation signals the need for ATR synthesis. ADR aUosterically activates isocitrate dehydrogenase, thereby increasing the rate of the citric acid cycle and the production of NADH and FADH. The elevated levels of these reduced coenzymes, in turn, increase the rate of electron transport and ATR synthesis. 

In isocitrate, there is a CHOH group that is available for oxidation via the coenzyme NAD+ and the enzyme isocitrate dehydrogenase. NADH will then be reoxidized via oxidative phosphorylation, and lead to ATP synthesis. The oxidation product from isocitrate is oxalosuccinate, a -ketoacid that easily... 

Isocitrate dehydrogenase catalyzes the NAD-dependent reduction of isocitrate to a-ketoglutarate. The dimeric enzyme is regulated via phosphorylation. Phosphorylation on SerllS leads to a complete inactivation of the enzyme. 

An understanding of the molecular basis for regulation of isocitrate dehydrogenase by phosphorylation was facilitated by X-ray crystallography of the phosphorylated enzyme in complex with isocitrate. The crystal structures of mutants of the enzyme in which SerllS had been exchanged for aspartate or glutamate were also solved (Hurley et al., 1990). The structure of the enzyme in complex with the substrate isocitrate revealed the phophorylation site to be localized near isocitrate. SerllS itself binds the substrate directly via a H-bond with the O of isocitrate (fig. 2.13). 

Wang, Koshland, D.E. The reversible phosphorylation of isocitrate dehydrogenase of Salmonella typhimurium. Arch. Biochem. Biophys., 218, 59-67 (1982)... 

Phosphorylation of an enzyme can affect catalysis in another way by altering substrate-binding affinity. For example, when isocitrate dehydrogenase (an enzyme of the citric acid cycle Chapter 16) is phospho-rylated, electrostatic repulsion by the phosphoryl group inhibits the binding of citrate (a tricarboxylic acid) at the active site. 

Phosphorylation in bacteria. A bacterial enzyme whose activity is controlled by phosphorylation is isocitrate dehydrogenase. Transfer of a phospho group to the - OH of Ser 113 completely inactivates the... 

JH Hurley, P Thorsness, V Ramalingham, N Helmers, DE Koshland Jr, RM Stroud. Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase. Proc Natl Acad Sci (USA) 86 8635-8639, 1989. 

RD Chen, JA Grobler, JH Hurley, AM Dean (1996). Second-site suppression of regulatory phosphorylation in Escherichia coli isocitrate dehydrogenase. Protein Sci 5 287-295, 1996. 

Four of the Krebs cycle reactions are considered irreversible citrate synthase, isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, and succinyl-CoA synthase. In two of these, COz is evolved. In one reaction, succinyl-CoA synthase, a substrate-level phosphorylation takes place, in which a high-energy compound, GTP, is generated. Note that in three of the reactions NADH is... 

X1B-X14), and NADP-linked isocitrate dehydrogenase SIS, S16). Numerous studies have been carried out with isolated mitochondria to identify the source of NADPH for 11 -hydroxylation by studying the effect of inhibitors and uncouplers of oxidative phosphorylation in the presence of different hydrogen donors (83, 19S, SIO, SIS, SI4-SSS). Part of this work was inconclusive since several complicating features in the metabolism of adrenocortical mitochondria were insufficiently taken into account such as secondary inhibitory effects of the substrates, inhibitors, or uncouplers used 83, SI4, SSO, SSS) the requirement of transport of substrates across the mitochondrial membrane SS3, SS4) and the possibility of intramitochondrial dismutation reactions SS3). 

The answer is b. (Murray, pp 182-189. Scriver, pp 1521-1552. Sack, pp 121-138. Wilson, pp 287-317.) Reducing equivalents are produced at four sites in the citric acid cycle. NADH is produced by the isocitrate dehydrogenase-catalyzed conversion of a-ketoglutarate to succinyl CoA and by the malate dehydrogenase-catalyzed conversion of malate to oxaloacetate. FADH, is produced by the succinate dehydrogenase-catalyzed conversion of succinate to fumarate. Succinyl CoA synthetase catalyzes the formation of succinate from succinyl CoA, with the concomitant phosphorylation of GDP to GTP... 

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